Performing enzymatic hydrolysis at high protein concentrations could result in a reduction of the amount of water and energy consumed during the production of hydrolysates. However, this benefit may be counterbalanced by a decreased efficiency of the process [6]. Indeed in this thesis, it has been shown that the overall hydrolysis rate as well as the DH reached after 2 hours was decreased with increasing substrate concentration, up to 30 % (w/v) WPI (chapters 2, 3 and 5). Besides a slower kinetics, increasing the protein concentration also results in the formation of gels. Before the formation of a gel, aggregates may be formed. The formation of aggregates has been studied at quite low concentrations of whey proteins during enzymatic hydrolysis by BLP (5 % (w/w)) [2] and hydrolysis of soy glycinin by chymotrypsin [7]. It has been shown that hydrolysis is hindered and eventually stopped by the formation of aggregates during hydrolysis [8]. In chapters 2 and 3, the formation of a gel during hydrolysis also hindered further hydrolysis and made it impossible to reach DH values higher than 15 % at the high protein concentrations. Because of the increase in viscosity and the formation of a gel, samples cannot be collected during hydrolysis. This hindered the complete description of the hydrolysis process at high protein concentrations. Such gelation has been previously shown to occur in a 20 % (w/v) WPI solution during hydrolysis by Alcalase [9]. Protease induced gelation has also been described for the enzyme BLP (Bacillus licheniformis protease) and has been mentioned to occur at concentrations as low as 2 % (w/v) α-lactalbumin [10] and for 12 % (w/v) WPI [11].
During data collection for chapter 2, the formation of viscous solutions leading to the formation of a gel was observed for hydrolysis of 30 % WPI by Alcalase (0.13 μL/mg protein) at initial pH 8.0 and 40 °C. Under these conditions, the hydrolysates form a gel after 2 hours of hydrolysis at a DH ±15 %. To confirm this visual observation an additional experiment is reported here. The viscosity of a 30 % (w/v) WPI solution during hydrolysis by Alcalase was monitored using a Rheometer Anton Paar MCR 501. Changes in complex viscosity were measured every 2 minutes for 6 hours. The temperature was kept constant at 40 °C during the analysis. The viscosity was constant in the first 5,000 seconds of hydrolysis. After that, the viscosity increased up to the
formation of a gel after 2 hours of incubation. During the viscosity measurement the pH was not controlled, which makes the conditions slightly different from the one used for the experiment in the pH-stat. (figure 2)
Figure 2. Viscosity of a 30 % (w/v) WPI solution during incubation with Alcalase.
It is important to mention that the viscosity is constant during the first 5,000 seconds of incubation while a slower rate of hydrolysis is observed from the first minutes of incubation onwards with increasing substrate concentration (chapter 2). This indicates that an increasing viscosity cannot be the reason for a lower rate of hydrolysis at
elevated substrate concentrations. There may be formation of soluble aggregates,
which might hinder the hydrolysis with increasing substrate concentration.
When 30 % (w/v) WPI is hydrolyzed by BLP at pH 8.0 and 40 °C, a gel is formed after the first minutes of the hydrolysis (chapter 3). This is the reason why the influence of substrate concentration could only be studied up to 10 % (w/v) for hydrolysis with BLP (chapters 3 and 5). (table 2) Alcalase is an a-specific enzyme, while BLP is a specific enzyme. That gels are obtained at two different time points or DH values during hydrolysis with the two enzymes is probably the result of the formation of different peptides.
Table 2. Influence of the hydrolysis conditions on the hydrolysates gelation at 30 % (w/v) WPI.
SDS: sodium dodecyl sulfate; LBG: Locust bean gum.
The formation of a gel is hindering further hydrolysis and it also makes it difficult to handle the hydrolysates obtained. To try to avoid the formation of a gel and to be able to collect more samples during the hydrolysis by BLP of 30 % (w/v) WPI, a few additional trials were performed, which are reported here. This was done by addition of
0.01 0.1 1 10 100 1000 10000 0 5000 10000 15000 20000 25000 Co m pl ex vi sc osi ty (P a. s) Time (s)
Enzyme pH Temp (°C) Additives Time of gelation
Alcalase 8.0 40 2 hours
BLP 8.0 40 5 min
BLP 8.0 40 1 % SDS 15 min
additives to the solution before hydrolysis. (table 2) First, urea was added to a final concentration of 2 M or 4 M to a 30 % (w/v) WPI solution before hydrolysis. While this was done to avoid gelation, the addition of urea resulted in the formation of a gel at the initial conditions of hydrolysis (pH 8.0 and 40 °C). Secondly, SDS to a final concentration of 1 % (w/v) was added to the solution before hydrolysis. This resulted in a slight delay in the formation of the gel. A further increase of the concentration of SDS to 2 % (w/v) resulted in inactivation of the enzyme. Locust bean gum (LBG), a neutral polysaccharide, has been used in chapter 2 to determine the influence of viscosity on the hydrolysis rate for 1 % (w/v) WPI solution. The same polysaccharide was also added to a final concentration of 0.1 % (w/v) to 30 % (w/v) WPI for hydrolysis by BLP. The increase in viscosity of the initial solution actually led to a postponed formation of the gel, after 50 minutes of hydrolysis.
In conclusion, the formation of a gel during the hydrolysis of 30 % (w/v) WPI by BLP can be slightly postponed in time by addition of chemicals. Still, in all cases gels were formed at the higher concentrations, showing that this is a principle problem that cannot be easily solved.
In addition to the gel formation during hydrolysis, deviations in the properties of the initial protein solutions were observed with increasing protein concentrations. This has been shown by determining the conductivity of protein solutions as a function of protein concentration. Above 10 % (w/v) WPI the correlation between conductivity and protein concentration deviates from linearity (chapter 2). Another deviation from linearity has been observed by measuring the fraction of free water by relaxation time using NMR. For concentrations of 20 and 30 % (w/v) WPI a different linear regime was observed than for protein concentrations of 0.1 to 10 % (w/v) WPI (chapter 3). This indicates a deviation from linearity, i.e. ideal behavior. Ideal systems are defined as systems in which the number of molecules is so low that they do not influence each other [12]. By increasing protein concentration, as seen by the conductivity, concentration dependent effects cannot be neglected. Such solutions are called non-ideal. One reported observation is the increase in protein stability in crowded systems. Molecular crowding is commonly used to describe the effect of increased stability of molecules in the interior of cells, where the total concentration of macromolecules is high (i.e. up to 400 g/L) [12]. This is experimentally studied by addition of a high concentration of a crowder to a solution of protein. Molecular crowding effects are also sometimes called excluded volume effects. It is generally assumed that the hydration structure of proteins is altered in the presence of crowder molecules, which are either small molecules (e.g. glycerol) or macromolecules (e.g. PEG) [13]. Based on the non-ideal behavior of conductivity and decreased hydrolysis, in combination with the theories of molecular crowding, it can be assumed that the hydration at concentrations as high as 30 % (w/v) WPI deviates from the hydration of proteins in a diluted (ideal) solution.
The observed gelling behavior of concentrated systems (at 30 % (w/v) WPI) seems to be a fundamental property of such concentrated systems and could not be suppressed or avoided by adding SDS or urea. For samples at lower concentrations, no gel was
formed, while still a deviation from ideality was noted. In conclusion, these examples show the difficulty in understanding and describing behavior of highly concentrated protein solutions due to their non-ideality.